Clinical Significance. Neuronal communication involves complex and coupled networks of different chemicals including neuropeptides, neurotransmitters, and signaling proteins called cytokines. Chemokines (chemoattractant cytokines) and cytokines act as immunoregulators and neuroregulators in the central nervous system (CNS) and are considered a third neurocommunication system. These important proteins affect many different disease states. A comprehensive understanding of the nervous system cannot be achieved without decoding this chemical signaling network. One of the primary barriers preventing decoding these chemical networks is the lack of appropriate chemical analysis tools that can be applied to study in vivo biochemistry in rodent models. In particular, microdialysis sampling of these important cytokines and peptides has been difficult to implement. Neuropeptide and cytokine ECF concentrations are generally unknown and their processing via receptor meditated uptake (cell-based receptors and for cytokines - soluble receptors) is poorly understood. The risks associated with performing this research are greatly offset by several high-impact benefits. 1) Significant biochemical information about the real protein concentrations and not just upregulated mRNA levels and their makeup (e.g., which cytokines) in the CNS will be gained. 2) True in vivo measurements will be made rather than post-mortem measurements. Experimental Approach. We hypothesize that modification to existing brain dialysis methods of either inclusion of a free antibody or antibodies immobilized to small microspheres will significantly improve protein relative recovery into dialysis probes that are implanted into the rat brain. Additionally, new flow regimes including recycled flow and flow reversal with new pumps are included as methods to increase relative recovery of the targeted proteins. Our primary focus will be on a select group of cytokines and neuropeptides related to epilepsy (cytokines {CCL2 [MCP-1], CCL3 [MIP-1a], CCL4 [MIP-12], CCL5 [RANTES], KC/GRO, IL-1b, IL-6, IL-10, and TNF-a} and neuropeptides {angiotensin IV, 2-amyloid, cholecystokinin, corticotropin releasing hormone, and neuropeptide Y). These multiplexed assays will allow for the simultaneous measurement of different peptides in volumes as low as 25 5L of dialysate collected from the rat brain. PUBLIC HEALTH RELEVANCE: Proteins called cytokines as well as other neuropeptides are known to affect different human diseases related to the brain including, alcoholism, anxiety, appetite, depression, epilepsy, multiple sclerosis, pain, sleep, and various psychiatric disorders. These peptides and proteins are difficult to measure in the living brain. This work seeks to develop analytical chemistry methods to collect and detect these proteins in rodents to allow for rapid translational medical treatments for humans.